Meiosis Drives Extraordinary Genome Plasticity in theHaploid Fungal Plant Pathogen MycosphaerellagraminicolaAlexander H. J. Wittenberg1,4.¤, Theo A. J. van der Lee1., Sarrah Ben M’Barek1,5., Sarah B. Ware1,5,
Stephen B. Goodwin2, Andrzej Kilian3, Richard G. F. Visser1,4, Gert H. J. Kema1*, Henk J. Schouten1
1 Plant Research International B.V., Wageningen University and Research Centre, Wageningen, The Netherlands, 2 United States Department of Agriculture (USDA)-
Agricultural Research Service (ARS), Crop Production and Pest Control Research Unit, and Department of Botany and Plant Pathology, Purdue University, West Lafayette,
Indiana, United States of America, 3 Diversity Arrays P/L, Yarralumla, Canberra, Australian Capital Territory, Australia, 4 Graduate School Experimental Plant Sciences,
Laboratory of Plant Breeding, Department of Plant Sciences, Wageningen University and Research Centre, Wageningen, The Netherlands, 5 Graduate School Experimental
Plant Sciences, Laboratory of Phytopathology, Department of Plant Sciences, Wageningen University and Research Centre, Wageningen, The Netherlands
Abstract
Meiosis in the haploid plant-pathogenic fungus Mycosphaerella graminicola results in eight ascospores due to a mitoticdivision following the two meiotic divisions. The transient diploid phase allows for recombination among homologouschromosomes. However, some chromosomes of M. graminicola lack homologs and do not pair during meiosis. Becausethese chromosomes are not present universally in the genome of the organism they can be considered to be dispensable.To analyze the meiotic transmission of unequal chromosome numbers, two segregating populations were generated bycrossing genetically unrelated parent isolates originating from Algeria and The Netherlands that had pathogenicity towardsdurum or bread wheat, respectively. Detailed genetic analyses of these progenies using high-density mapping (1793 DArT,258 AFLP and 25 SSR markers) and graphical genotyping revealed that M. graminicola has up to eight dispensablechromosomes, the highest number reported in filamentous fungi. These chromosomes vary from 0.39 to 0.77 Mb in size,and represent up to 38% of the chromosomal complement. Chromosome numbers among progeny isolates varied widely,with some progeny missing up to three chromosomes, while other strains were disomic for one or more chromosomes.Between 15–20% of the progeny isolates lacked one or more chromosomes that were present in both parents. The twohigh-density maps showed no recombination of dispensable chromosomes and hence, their meiotic processing mayrequire distributive disjunction, a phenomenon that is rarely observed in fungi. The maps also enabled the identification ofindividual twin isolates from a single ascus that shared the same missing or doubled chromosomes indicating that thechromosomal polymorphisms were mitotically stable and originated from nondisjunction during the second division and,less frequently, during the first division of fungal meiosis. High genome plasticity could be among the strategies enablingthis versatile pathogen to quickly overcome adverse biotic and abiotic conditions in wheat fields.
Citation: Wittenberg AHJ, van der Lee TAJ, Ben M’Barek S, Ware SB, Goodwin SB, et al. (2009) Meiosis Drives Extraordinary Genome Plasticity in the HaploidFungal Plant Pathogen Mycosphaerella graminicola. PLoS ONE 4(6): e5863. doi:10.1371/journal.pone.0005863
Editor: Jason E. Stajich, University of California, Berkeley, United States of America
Received December 5, 2008; Accepted March 27, 2009; Published June 10, 2009
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the publicdomain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: The US Department of Energy - Joint Genome Institute provided DArT sequences. The HudsonAlpha Institute for Biotechnology provided support ingenome assembly. The Netherlands Genomics Initiative (grant 050-72-401) supported AHJW with a one-year fellowship. The Sixth Framework Programme’s(BioExploit-EU FP6) Food Quality and Safety priority (contract No. 513959) supported GHJK, TAJL and SBM. SBM is a recipient of a UNESCO-L’Oreal fellowship. Thefunders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
¤ Current address: Keygene N.V., Wageningen, The Netherlands
. These authors contributed equally to this work.
Introduction
Fungi provide attractive model systems to analyze processes that
occur during meiosis. Many fungi are haploid, which greatly
simplifies genetic studies. Furthermore, complete recovery of the
meiotic products, or tetrads, is possible in ascomycete fungi, and
these tetrads can be analyzed for the segregation of genetic
markers. Tetrad analyses of Aspergillus nidulans and Neurospora crassa
have been instrumental in answering fundamental questions
concerning meiosis [1–3]. Here we describe genetic studies in
another filamentous ascomycete, Mycosphaerella graminicola (asexual
stage: Septoria tritici). This fungus causes septoria tritici blotch (STB)
of wheat, a disease characterized by necrotic blotches on the
foliage. These blotches contain asexual (pycnidia) and sexual
(pseudothecia) fructifications. M. graminicola represents an intrigu-
ing model for fundamental genetic studies of plant-pathogenic
fungi. Field isolates of this pathogen usually have 18–21
chromosomes, the highest number reported among ascomycetes.
Furthermore, these chromosomes have an extraordinary size
range, varying from 0.39 to 6.09 Mb [4]. Genome plasticity -
comprising processes such as inversions, deletions, insertions and
translocations that translate into chromosome length polymor-
phisms (CLPs) as well as chromosome number polymorphisms
(CNPs) - results in a genome size that varies between 32 and
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40 Mb, similar to other filamentous ascomycetes [4–9]. M.
graminicola has an active sexual cycle under natural conditions,
which is an important driver of STB epidemics and results in high
genetic diversity of populations in the field [10–12].
Analyses of a cross between two M. graminicola strains that
originated from bread wheat fields in The Netherlands resulted in
the first genetic linkage map of a Mycosphaerella species [13,14].
Although this map was a major milestone, the anonymous AFLP
and RAPD markers complicated integration of genetic data sets.
In addition, the number of markers was limited and the map
resolution was too low to assess the complications anticipated
during meiosis due to the CLPs and CNPs commonly observed
among M. graminicola isolates [9].
The exact origin and maintenance of CNPs and CLPs are not
known. A likely hypothesis is that they can be generated or lost
during meiosis. Recombination between chromosomes that differ
in length could give rise to derivatives with CLPs [15].
Nondisjunction during meiosis I or II would generate CNPs. To
test these hypotheses, we used the recently developed Diversity
Arrays Technology (DArT) for the first time on a haploid fungal
genome [16–20]. The parallel genotyping of progeny isolates using
several thousands of DNA fragments spotted on a microarray and
subsequent analysis resulted in one of the most dense genetic
linkage maps currently available for a fungus. This enabled high-
resolution genetic linkage analyses to study the meiotic processing
of CNPs and CLPs as well as the generation of new genome
plasticity in M. graminicola. We frequently observed the loss of one
or more chromosomes, disomy and translocations. This extraor-
dinary genome plasticity helps to explain the high genetic diversity
observed within natural populations of this fungus and most likely
facilitates rapid adaptation to changing environments.
Materials and Methods
Fungal isolates and DNA extractionWe used three isolates of M. graminicola: IPO323 and IPO94269
were isolated from bread wheat in the Netherlands and IPO95052
was isolated from durum wheat in Algeria. Isolate IPO323 was
crossed to both IPO94269 and IPO95052 using a previously
developed in planta protocol [10], resulting in 68 and 148 progeny,
respectively. All progeny isolates were collected and analyzed
individually. DNA of parents and progeny was isolated using the
Wizard Genomic DNA purification kit (Promega Madison, WI),
starting with approximately 10 mg of lyophilized spores. Tables S1
and S2 provide an overview of the progeny isolates used in this study.
DArT procedureGeneration of genomic representations, library construction,
target preparation and image analysis were essentially performed
as described previously [17,18], with the modifications described
by Wittenberg et al. [16]. The adapter and primer oligonucleotide
sequences used in this study are listed in Table S3. For details see
Text S1.
Nomenclature of markersAFLP markers were designated by the primer combination used
for the amplification and the approximate length of the generated
fragment [14]. For both AFLP and DArT markers the prefix A or B
indicated the phase of the marker; those originating from parent
IPO323 had the prefix A while markers from parent IPO95052
were indicated by the prefix B. DArT markers identified in cross
IPO3236IPO94269 originating from isolate IPO95052 could be
assigned the prefix A or B, as IPO94269 was not used for the library
construction. Markers segregating in both populations received the
prefix C. In addition, DArT markers were designated by the
enzyme combination used for complexity reduction (BamHI, MseI
and RsaI: BMR or HindIII, MseI, RsaI: HMR), the 384-well plate
number and the position of the fragment in that plate (i.e.,
AHMR_04I09). Recently, 23 SSR loci were identified in M.
graminicola, 21 of which could be positioned on the existing linkage
map along with two previously published SSR loci [21,22]. The
newly generated DArT markers were used to integrate the new
IPO3236IPO94269 map with the existing map of that population
[14]. Moreover, six of these SSRs also differentiated the parents of
the second mapping population. To enable the mapping of these
SSRs in the IPO3236IPO95052 progeny, amplification reactions
were performed as described by Goodwin et al. [21].
Selection of unique segregation patterns and merging oftwin isolates
The binary scores of polymorphic markers were converted to
the correct allelic phase based on the scores of the parents. A Perl
script was written that grouped loci with identical segregation
patterns after disregarding unknown scores. The marker with the
highest call rate (percentage of scored individuals) was selected as a
representative for each group. The script also calculated the call
rate for each individual genotype and the global call rate for the
whole dataset. Individual genotypes were incorporated into the
scoring table when at least 95% of the grouped markers could be
scored. In M. graminicola, twin progeny isolates arise from the
mitotic division that follows meiosis II in the ascus, resulting in
four pairs of genetically identical ascospores. Although the
random-ascospore progenies that resulted from the crossing
protocol minimized the isolation of twin isolates, the large number
of markers identified identical progeny efficiently. These were used
to calculate the reproducibility of the different marker types and
were merged before the mapping analyses.
Construction and comparison of the linkage mapsThe genetic linkage maps of the individual crosses as well as the
bridge map were constructed with the software package JoinMap
3.0 [23]. A detailed description of the mapping process for the
individual maps is given in Text S1. The use of IPO323 in both
crosses enabled the efficient generation of an integrated bridge map
of the M. graminicola genome. The bridge map was used to compare
the order of the loci in the constructed IPO3236IPO94269 and
IPO3236IPO95052 maps. We used MapChart 2.2 [24] for the
graphical representation of the genetic linkage maps.
Evaluation of loss or gain of chromosomesWe used graphical genotyping to compare the marker scores (A
or B) and the phase (A or B) of the markers, which enabled us to
identify whether each marker was present or absent in a particular
progeny isolate. In cases where a linkage group (LG) was
constructed from both marker types and a specific progeny isolate
lacked all of these markers, we concluded that the isolate missed
that LG. In cases where a LG was constructed from both marker
types and a specific progeny isolate was scored present for all
markers, we concluded that the isolate had an extra copy, derived
from the other parent, of that particular LG. Hence, chromosome
polymorphisms in progeny isolates were determined in silico if A
and B markers that were assigned to a specific LG were always
absent or present in a particular progeny.
PCR verification of loss and gain of chromosomesDNA samples of the parental isolates (IPO323, IPO94269 and
IPO95052), progeny isolates that showed absence of specific LGs
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by graphical genotyping (Table S4) and two control progeny
without these aberrations were used as templates in the PCR
reactions. PCR was performed using SSR markers and specific
primer pairs developed from the sequenced DArT markers located
on the missing linkage groups (Tables S5 and S6). To assure that
absence of an amplicon was not caused by PCR failure, a positive
PCR control was included that should be present in all parents and
progeny that were tested. The SSR marker loci ac-0007 (LG8) and
gga-0001 (LG12) were amplified in combination with the PCR
control SSR locus ag-0003 (LG2). For the amplicons derived from
the DArT marker sequences, the DArT fragments
CABMR_07D07 (129 bp; LG1) or AHMR_08O09 (728 bp;
LG15) served as positive PCR controls.
PCR reactions were performed in a total volume of 20 ml
containing 20 ng of genomic DNA, 16PCR buffer (Roche), 1 ml
of each of the forward and reverse primers used as a control
(2 mM), 2 ml of each forward and reverse primer (2 mM), 0.8 ml of
dNTPs (5 mM) and 0.2 ml of Taq DNA polymerase (5 U/ml).
Amplification conditions were as follows: 94uC for 2 min, 12
cycles of 94uC for 30 sec, 66uC for 30 sec minus 1uC per cycle,
72uC for 30 sec; 27 cycles of 94uC for 30 sec, 53uC for 30 sec,
72uC for 30 sec; 72uC for 7 min, followed by a cooling-down step
to 10uC. The SSR amplicons were separated on 6% non-
denaturating acrylamide gels using a Mega-Gel Dual High-
Throughput Vertical Electrophoresis Unit (CBS Scientific, Del
Mar, California, USA). Amplicons based on the DArT sequences
were separated on 2.5% agarose gels.
Results
Marker selection and qualityAmong the 68 progeny isolates from the M. graminicola
IPO3236IPO94269 cross, 1042 new DArT markers were
obtained. The DArT markers were added to the first genetic
linkage map of M. graminicola [14], consisting of 271 AFLP
markers, 57 RAPD markers and two markers for the biological
traits avirulence (Avr) and mating type (mat). Twenty-five SSR
markers also were added to the combined linkage map (Table S7)
[21]. For the 148 progeny isolates of the M. graminicola
IPO3236IPO95052 cross, 1154 DArT markers were obtained
that were combined with six SSR markers and the markers for the
two biological traits (Table S8). After analysis of the marker data,
31 twins were detected in the M. graminicola progenies (Table S9).
These twins result from the mitotic division that follows meiosis II
in the ascus. The twin data enabled the dissection of mitotic or
meiotic events that drive the generation of CLPs and CNPs.
Eventually, the merged scoring tables comprised 60 individuals for
the IPO3236IPO94269 cross and 125 individuals for the
IPO3236IPO95052 cross (Table S10). Because twins can be
regarded as biological replicates, they also were used to evaluate
the reproducibility of the marker scores for the different marker
technologies. In our study, DArT and AFLP markers appeared to
be more reproducible than the RAPD markers. Therefore, RAPD
markers were excluded to improve the quality of the maps.
Although the reproducibility for both DArT and AFLP was very
high, the frequency of double crossovers in the final maps was
much lower for DArT than for AFLP markers (0.24% compared
to 0.96%), indicating the superior reliability of the DArT markers.
Construction and comparison of the linkage mapsThe combined genetic linkage maps contain 2078 markers
comprising 1793 DArT, 258 AFLP, and 25 SSR DNA markers,
plus the two markers that co-segregate with the biological traits
Mat and Avr (Table S11). The grouping and the order of the
markers in the M. graminicola IPO3236IPO94269 cross were
highly similar to those in the previous maps [14,21]. Compared to
the previous map both new maps span a considerably larger part
of the genome. In both crosses close to 99% of the segregating
markers were reliably positioned, indicating that the current
genetic linkage maps cover the complete genome.
The new genetic linkage map of the IPO3236IPO94269 cross
is 638 cM longer than the first linkage map, and spans 1854 cM
with 1317 markers on 451 unique map positions, with an average
distance of 4.1 cM between the markers (Table S12). Nearly all
markers (98.2%) were positioned on 24 LGs. Some of the smaller
LGs that were observed in the first map merged with other LGs
[14]: 10 LGs in the first map merged into five larger LGs, while six
small new LGs were formed. For example, LGs 3 and 4 in the first
map merged with LGs 22 and 17, respectively, in the new map.
The order of the AFLP markers in the first and new map remained
similar, although more AFLP markers were positioned in the latter
(223 vs. 258 out of 271, representing 82.3% and 95.2%,
respectively). The genetic linkage map of the M. graminicola isolate
IPO3236IPO95052 cross spans 1946 cM and contains 1144
markers on 486 unique map positions on 23 LGs (comprising
98.5% of the generated markers), with an average distance of
4.0 cM between the markers (Table S12).
We also constructed a bridge map to compare the individual
linkage maps using markers that segregated in both mapping
populations. The resulting integrated map spans 1435 cM (,75%
of both individual maps) and contains 372 markers on 251 unique
map positions. A total of 22 LGs from each of the individual
crosses was aligned with the bridge map, and the marker order was
similar to those on the two individual genetic maps (Figure 1 and
Figure S1). The 21 LGs in the bridge map is close to the estimated
number of chromosomes based on electrophoretic and cytological
karyotyping [4,14] and is identical to the number of chromosomes
of the finished genome sequence (http://genome.jgi-psf.org/
Mycgr3/Mycgr3.home.html) (Table S13).
TranslocationsWe identified eight DArT markers that were positioned very
differently in the two maps, which is indicative of translocations.
They represented five translocations between isolate IPO323 and
either IPO94269 or IPO95052 and involved four inter-LG and
one intra-LG translocations (CBBMR_14G17 in LG 6) (Table
S14). Another translocation between IPO323 and IPO94269
involved an SSR locus [21] that segregated in a diploid fashion in
the isolate IPO3236IPO94269 cross (1:1:1:1 ratio, x2 = 1.25,
0.25,P,0.75) and was mapped on LG 21 in IPO323 and on LG
4+17 in IPO94269. In addition, we obtained indications for a
possible larger translocation involving LG F (Figure S1).
Meiosis drives extraordinary genome plasticityParental CNPs. LGs 21 and C in the M. graminicola
IPO3236IPO94269 cross span less than 2 cM and contain 21
and 36 markers (AFLP, SSR and DArT), respectively.
Interestingly, all of these markers are inherited from isolate
IPO323. This suggests that these two LGs are present in IPO323
but absent in isolate IPO94269. In the progeny of the
IPO3236IPO95052 cross these linkage groups do show
recombination, which resulted in much larger genetic distances
of 21 cM and 24 cM, respectively. These results indicate that both
linkage groups are present in isolates IPO323 and IPO95052, but
are absent in IPO94269. An example of the difference in
recombination frequency is shown for LG 21 in Figure S2.
Meiotic transmission of CNPs. Graphical genotyping allows
the tracing of the genetic make up of progeny isolates. Among the
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progeny of the M. graminicola IPO3236IPO95052 cross, LGs that
were regularly absent either individually or in combination included
LGs 8, 12, 13, 15, 21, A, B and C. LGs 21 and C are absent in
IPO94269, and frequently were missing in the M. graminicola
IPO3236IPO94269 progeny along with LGs 8, 12, 13 and A that
were also often missing in this progeny (Table S4). In these cases LGs
present in both parents were absent in one or more progeny isolates
(Figure 2). We also observed a progeny isolate (#40) from the M.
Figure 1. Co-linearity of genetic linkage maps for Mycosphaerella graminicola crosses IPO3236IPO95052 (left) andIPO3236IPO94269 (right) with a bridge map (middle) generated with markers that segregated in both crosses. Common markersare shown in bold and start with the prefix C, SSR markers are shown in blue and markers that are translocated in red. DArT markers were namedaccording to phase of the marker (A = IPO323, B = IPO95052 or IPO94269), complexity reduction method used (BMR or HMR), and location in thespotting plate (e.g. BBMR_15L11). LG and AFLP nomenclature is according to Kema et al., 2002. Segregation distortion of the markers is indicatedwith * (P,0.05), ** (P,0.01), *** (P,0.005) or **** (P,0.001).doi:10.1371/journal.pone.0005863.g001
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graminicola IPO3236IPO94269 cross that contained all markers from
both parents on LG 13, indicating that this isolate was disomic for this
relatively small chromosome (577 kb). In the same progeny set we
identified another isolate (#51) that was disomic for LG 1, which
represents one of the largest chromosomes (3.26 Mb) in the genome
of M. graminicola isolate IPO323. If nondisjunction occurs during
meiosis I, two paired chromosomes are pulled to one cell leading to
loss of that chromosome in the other cell (Figure 2B). In this case, one
haploid M. graminicola isolate would become heterozygous disomic for
that chromosome. If nondisjunction occurs during meiosis II, two
sister chromatids are not divided between the two cells but are both
pulled to the same cell (Figure 2C). This evidently leads to two
identical copies of the chromosome in that cell, and hence to
homozygous disomy in one cell and to absence in the other cell.
Unfortunately, homozygous disomy could not be detected with the
techniques used for our analysis.
Twins do not show CNPs. The large number of markers
permitted easy identification of identical progeny and allowed
determination of the stage at which CNPs were generated. In total,
we detected 31 twins in the M. graminicola progenies, whose identity
was visualized by graphical genotyping (Table S12). In four cases
we could demonstrate that LGs that were present in both parents
were absent in both isolates of a twin pair (Table S15). This is
illustrated for twin pair 2137–2139 in Figure 2D.
PCR confirmation. The observed aberrations and graphical
genotyping analyses were confirmed by PCR assays (Figure 2D, E).
Additional SSR and PCR assays confirmed the graphical
genotyping results for six out of eight LGs. The absence of two
LGs was confirmed by scoring of co-dominant SSR markers that
are located on LG 8 (ac-0007) and LG 12 (gga-0001). In all progeny
isolates that lacked these LGs, none of the parental alleles was
amplified (Figure S3A). The absence of LGs 8, 12, 13, 15, A and C
was further confirmed by diagnostic PCR analysis (Table S5) for the
mapped DArT markers. Indeed, none of these markers was
amplified in the progeny isolates that, according to the graphical
genotyping, lacked these particular LGs (Figure S3B and S3C).
However, amplicons of the expected size were always generated
from the relevant checks, i.e., parental isolates, two progeny isolates
that inherited the LG normally and a PCR amplification control.
To confirm the disomy for LG 1 in progeny isolate #51, we
performed PCR assays based on deletion polymorphisms
(Figure 2E, Table S6) identified by comparative analyses of
IPO94269 BAC-end sequences with the draft genome sequence
(v.2.5) of IPO323. These PCRs confirmed the graphical
genotyping results indicating that a series of progeny isolates lost
one or more complete chromosomes, while other isolates received
an extra copy of a particular chromosome.
In summary, the high-density mapping enabled the detection of
meiotically driven and frequently occurring CNPs and CLPs in
sexual progenies of the haploid plant pathogen M. graminicola. We
identified 42 isolates that showed loss of a linkage group that was
present in both parents compared to only two disomic isolates.
Progenies showed 15 and 20% CNPs compared to the parents in
the IPO3236IPO94269 and IPO3236IPO95052 crosses, respec-
tively. Interestingly, the chromosomes lost were the same in both
populations (Table S4). We performed 17 additional backcrosses
and F2 crosses between progeny isolates that showed substantial
CNPs. All crosses except one were successful and resulted in viable
progeny (Table S15).
Discussion
The genome of M. graminicola is highly plastic, based on the
detailed analyses provided by the high-density genetic linkage
maps. Eight chromosomes were missing in one or more progeny
and can be considered dispensable, while other chromosomes
occasionally were disomic. As many as three chromosomes were
missing from individual progeny isolates, with no apparent effect
on fitness. As expected, much of the genome plasticity is generated
during meiosis and this could help to explain the high adaptability
observed in field populations of this pathogen.
Dispensable chromosomes have been found in other fungi but
they usually occur at a low frequency and typically represent single
or a few chromosomes. For example the plant-pathogenic fungi
Alternaria alternata, Cochliobolus heterostrophus, Leptosphaeria maculans,
Magnaporthe grisea and Nectria haematococca as well as the insect
pathogen Metarhizium amisopliae each had only a single chromo-
some that was dispensable [25–30]. Dispensable chromosomes in
these species usually contain genes involved in pathogenicity or
virulence [27,28,30,31], whereas in others they don’t [32]. In M.
graminicola, genes involved in host plant perception did not map to
any of the eight identified dispensable chromosomes [33]. Hence,
the function of genes on dispensable chromosomes in M. graminicola
is yet unknown.
Genome instability is a major cause of disorders, and a range of
genes has been identified that have a role in maintaining genome
integrity [34]. In addition, polyploidy and aneuploidy are
considered evolutionary pathways to reproductive isolation and
speciation [35,36]. The mitotic and meiotic pairing and
transmission of homologous chromosomes with length polymor-
phisms has been studied intensively in models such as the fungi
Saccharomyces cerevisiae, N. crassa and Coprinus cinereus [15,37,38].
These model systems have substantially increased our knowledge
of meiotic processes [39], but they mostly involved cytogenetic
Figure 2. Nondisjunction during meiosis in the haploid fungus Mycosphaerella graminicola results in chromosome numberpolymorphisms due to the loss or gain of specific chromosomes. A. Meiosis starts with the merging of nuclei from two different strains,leading to a transitory diploid cell. Karyogamy is followed immediately by meiosis I and II, resulting in four haploid cells. These four cells areduplicated during a subsequent mitotic step, leading to eight ascospores per ascus. Each ascospore is genetically identical to one other ascosporewithin the same ascus. Such pairs of identical ascospores are called twins. We identified several twins in progenies of M. graminicola. When a strain ofa descendant lacked one or more chromosomes, the twins originating from the first mitotic cell division after meiosis always appeared to lack thesame chromosomes. This indicates that chromosomes are stable during mitosis but can be lost during meiosis. B. Chromosome loss during meiosiscan be a result of failure of separation of homologous chromosomes during meiosis I, or C. of the failure of separation of sister chromatids duringmeiosis II. D. Graphical genotyping of LG 8. The chromosomal segments descending from IPO323 are rendered in red, and the segments fromIPO95052 in blue. Markers are scored as present (black) or absent (white). As the marker scores on all linkage groups were identical for these twoisolates, we concluded that the descendants 2137 and 2139 are twins. However, both isolates lack all markers located on LG 8. This is a clearindication of absence of this linkage group in these isolates. Strikingly, this linkage group is present in both parents. For further verification, sevenDArT markers spanning the length of LG 8 were converted into simple PCR markers. In addition, one SSR marker was used. All markers appeared to beabsent in the twin isolates 2137 and 2139. This confirms the absence of LG 8 in these twins and indicates nondisjunction during meiosis as the cause.E. Nondisjunction not only results in loss of a chromosome in one twin but also to disomy for that chromosome in another twin from the same ascus.The graphical genotyping of isolate #51 illustrates heterozygous disomy for LG 1, which was confirmed by a PCR screen for deletion markers thatunequivocally showed the presence of two copies of this chromosome in this haploid fungus.doi:10.1371/journal.pone.0005863.g002
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studies and mutant strains [40,41]. A high-density genetic linkage
map provides a strong genome-wide alternative for precise
analyses of meiosis. However, the number of high-density genetic
maps for fungi is limited due to difficulties and costs of high-quality
marker generation and scoring required for their generation [42].
Here, we report the meiotic processing and generation of genomic
plasticity using a high-density genetic linkage map for M.
graminicola. This unusual approach enabled the detection of
Mendelian and non-Mendelian inheritance patterns and elucidat-
ed the underlying meiotic principles that frequently resulted in
progeny with CNPs.
It is very clear that meiosis not only maintains but also drives
novel CNPs in M. graminicola, which most likely result from
nondisjunction during the second meiotic division. We noticed
that 15–20% of progeny isolates were missing one or more
chromosomes that were present in the two parents. Interestingly,
the same chromosomes were dispensable in both crosses. PCR
analyses confirmed most of the CNPs, including the disomic
chromosomes. Despite graphical genotyping indications for the
absence of LGs 21 and B in the M. graminicola IPO3236IPO95052
progeny, PCR amplifications with several primer combinations
derived from the mapped DArT markers on these LGs were
inconclusive, although BLAST analyses to the genome of IPO323
revealed that they are single copy. The cause is unknown but may
be due to the high repetitive content of these LGs (not shown).
The high number of markers on the current linkage map
enabled accurate identification of twin isolates. These originate
from the mitotic division after meiosis and provided a unique
opportunity to test the meiotic origin of CNPs. If CNPs resulted
from aberrations during mitosis, twin isolates would show
differences in chromosome number and could not have been
identified. In M. graminicola, we repeatedly observed the loss of the
same chromosome in both twin isolates, which demonstrates it was
lost during meiosis and that CNPs are mitotically stable. We
cannot exclude the possibility of occasional mitotic instability
between isolates that otherwise would have been identified as
twins, but if it occurs it appears to be very rare. Hence, we
conclude that CNPs in M. graminicola are driven by meiosis.
Nondisjunction during either meiotic division results in progeny
with CNPs due to gains or losses of entire chromosomes. However,
the number of CNPs is twice as high after nondisjunction during
meiosis I compared to meiosis II. Moreover, besides chromosome
loss, meiosis I results in heterozygous and meiosis II in
homozygous disomy. Crossovers may result in heterozygozity for
part of the chromosome only, but the dispensable chromosomes
are small so crossovers occur less frequently. Our data revealed
frequent loss of chromosomes, but we only rarely observed
heterozygozity. This indicates that nondisjunction occurred
preferentially during meiosis II. Unfortunately, our marker
technology did not enable the quantitative determination of copy
numbers to confirm homozygous disomy.
Meiotic processing of CNPs in other fungi varies. For the related
ascomycete Leptosphaeria maculans, twin genotypes were also always
identical in respect to the presence or absence of a dispensable
chromosome [25]. This indicates that, similar to M. graminicola, the
dispensable chromosome in L. maculans is mitotically stable.
However, in the evolutionarily more distantly related ascomycete
Magnaporthe oryzae [26], presence of a dispensable chromosome
varied in twin isolates, indicating that mitotic transmission of
dispensable chromosomes may be unstable in some ascomycetes.
Apart from these differences and the fact that M. graminicola has
up to eight dispensable chromosomes, a most striking aspect is that
the widespread CNPs - involving multiple chromosomes - in M.
graminicola do not hamper sexual reproduction. Interestingly, one
of the factors inhibiting female fertility in M. grisea is present on a
dispensable chromosome [32]. We do not have such evidence for
M. graminicola. Recent karyotyping experiments showed that isolate
IPO323 has at least two additional chromosomes compared to
IPO94269 [4]. Nevertheless, we were successful in crossing these
two isolates and made 17 additional crosses between M. graminicola
isolates that showed substantial CNPs. Chromosomes without a
homologous partner cannot pair, will have zero recombination
and might be expected to be lost during meiosis. However, our
data indicate that in M. graminicola they are normally transmitted to
progeny without distortion of the segregation ratio. For example,
in the progeny of the IPO3236IPO94269 cross, 34 and 35 out of
60 isolates contained the dispensable LGs 21 and C, respectively.
This shows that the CNPs present between the parents are
maintained during meiosis and are transmitted to approximately
50% of the progeny. Neither LG showed evidence of recombina-
tion as indicated by zero genetic distance between markers. The
segregation of the unique IPO323 markers on these LGs
confirmed the results of previous karyotyping experiments, that
individual dispensable chromosomes are transmitted intact
through meiosis [4]. This may well be among the first examples
of distributive disjunction in fungi, a process that involves
separation and distribution of non-recombining or non-homolo-
gous chromosomes during meiosis that is commonly observed in
Drosophila. In fungi distributive disjunction was shown in S. cerevisiae
by crossing strains that were monosomic for non-homologous
chromosome I and III [43]. In M. graminicola monosomic strains do
not occur as the fungus is haploid, but the dispensable
chromosomes were shown to segregate regularly. In S. cerevisiae
distributive disjunction is considered to be extremely rare as
monosomy does not frequently occur [43]. In M graminicola, it
might be essential as this study shows that CNPs occur frequently
and are generated during meiosis. It is unknown whether
distributive disjunction in M. graminicola also complies with the
physical interactions between non-homologous chromosomes as
was observed in S. cerevisiae [44].
In contrast, all LGs in the entire progeny set of the
IPO3236IPO95052 cross contain markers from both parents,
indicating that all parental chromosomes have homologous
partners. Hence, in this respect the differences between the two
Dutch bread wheat isolates (IPO323 and IPO94269) seem to be
larger than were those between IPO323 and the Algerian durum
wheat isolate IPO95052, underscoring the extraordinarily large
genetic differences within local populations of M. graminicola
[12,45].
CLPs have been observed in at least 37 fungal species and hence
seem to be a common feature of fungal genomes [15]. Clearly,
recombination between homologous chromosomes of unequal
length can result in new chromosome size variants. Moreover, the
pairing of repeated sequences, for instance resulting from
transposons, on different chromosomes during meiosis may lead
to translocations that may be an important cause of CLPs as
opposed to CNPs [38]. Subtelomeric variable regions such as those
in M. grisea are also a potential source of meiotically driven CLPs
[46]. The observed translocations in this study, as well as those in
previous analyses [8,9,47,48], most likely are responsible for the
widespread CLPs in the genome of M. graminicola [4,8,9].
Compared to CLPs, CNPs in other fungi are observed less
frequently, have not been analyzed through a map-based
approach, and are generally highly unstable. For instance, a
minichromosome in M. grisea showed non-Mendelian inheritance,
which was also observed in L. maculans whenever one parent
missed such a chromosome [25,32]. Crosses between L. maculans
isolates that both carried this minichromosome resulted in CLPs
Fungal Genome Plasticity
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[25]. Duplication of large chromosomal fragments in S. cerevisiae
occasionally results in the formation of supernumerary chromo-
somes that are highly unstable during mitosis [36,37]. In the
usually haploid human pathogen Cryptococcus neoformans, CNPs
occur frequently in diploid AD serotypes as a potential mechanism
to overcome slow filamentous growth [49] and, more recently,
CNPs were discovered resulting from the generation and
subsequent breakage of a dicentric chromosome [50]. CNPs in
haploid filamentous fungi such as N. crassa are generally either
lethal or seriously impair the sexual phase [38]. Diploid and
disomic isolates of N. crassa, originating from nondisjunction at
meiosis I, are highly unstable and do not differ in rates and
mechanisms of haploidization and mitotic crossing over [51].
Similarly, disomic strains in A. nidulans that resulted from
nondisjunction in meiotic metaphase I also were vegetatively
unstable [52,53].
In contrast to other species, CNPs in M. graminicola are
vegetatively stable. We hypothesize that the extraordinarily high
chromosome number of the M. graminicola genome [4] may
influence the frequency and fate of CNPs. The genome of M.
graminicola (39.8 Mb) is in the same size range as those of
Magnaporthe oryzae (41.6 Mb), Fusarium graminearum (36.5 Mb), A.
nidulans (30.0 Mb) and N. crassa (39.2 Mb). However, the number
of chromosomes in these fungi (N = 8, 4, 7 and 7, for A. nidulans, F.
graminearum, M. oryzae, and N. crassa, respectively) is much lower
than in M. graminicola (N = 21). Hence, loss of entire chromosomes
in these organisms may be lethal due to the presence of essential
genes. M. graminicola has the highest chromosome number and the
smallest autosomes in filamentous ascomycetes [4]. The present
study has revealed that M. graminicola also has the highest number
of dispensable chromosomes that vary from 0.39 to 0.77 Mb,
representing up to 38% of the chromosomal complement and
approximately 12% of its genome size. The frequent loss of
chromosomes in M. graminicola without noticeable effect on fitness
may be due to their small size. Dispensable chromosomes in many
other fungi carry functional genes that play an important role in
host-pathogen interactions [27–29,31,54,55]. In M. graminicola, loci
controlling host-pathogen interactions were not mapped on
dispensable chromosomes and substantial CNPs in progeny
isolates - up to three chromosomes per isolate covering as much
as 1.59 Mb - neither reduced pathogenicity nor sexual compat-
ibility [14,33]. Therefore, pathogenicity in M. graminicola does not
appear to be influenced by dispensable chromosomes.
In summary, our map-based approach is unique in analyses of
genomic plasticity and demonstrates that CNPs in M. graminicola
are meiotically generated and occur at much higher frequencies
than reported previously for any ascomycete. These aberrations
were observed in two crosses between field strains [10]. Since the
sexual cycle occurs continuously under field conditions it is likely
that meiotically driven CNPs play an important role in the high
level of genetic diversity [11,12,56] observed among isolates of M.
graminicola. The total genome content of M. graminicola isolates
varies between 32–40 Mb and each field isolate represents a
unique karyotype [4,9]. In this study we showed that in addition to
CLPs resulting from translocations, CNPs originate from aberra-
tions during meiosis, mostly by nondisjunction during meiosis II.
We hypothesize that the plasticity of the M. graminicola genome, as
characterized by its large and flexible set of dispensable
chromosomes, plays an important role in yet unknown processes
of adaptation. This is currently being addressed in a M. graminicola
crossing program aiming at individuals with a minimal genome
size that is devoid of any dispensable chromosome. Backcrosses of
such individuals with parental isolates will enable the selection of
progeny with individual dispensable chromosome additions. Such
a set will contribute significantly to understanding the role of
dispensable chromosomes in the life strategy of M. graminicola.
Supporting Information
Text S1 Supplementary text
Found at: doi:10.1371/journal.pone.0005863.s001 (0.06 MB
DOC)
Figure S1 Co-linearity of genetic linkage maps for Myco-
sphaerella graminicola crosses IPO3236IPO95052 (left) and
IPO3236IPO94269 (right) with a bridge map (middle) generated
with markers that segregated in both crosses. Common markers
are shown in bold and start with the prefix C, SSR markers are
shown in blue and markers that are translocated in red. DArT
markers were named according to phase of the marker
(A = IPO323, B = IPO95052 or IPO94269), complexity reduction
method used (BMR or HMR), and location in the spotting plate
(e.g. BBMR_15L11). LG and AFLP nomenclature is according to
Kema et al., 2002. Segregation distortion of the markers is
indicated with * (P,0.05), ** (P,0.01), *** (P,0.005) or ****
(P,0.001).
Found at: doi:10.1371/journal.pone.0005863.s002 (0.21 MB
PDF)
Figure S2 Alignment of linkage group 21 between the
IPO3236IPO95052 cross (left) and the IPO3236IPO94269 cross
(right) shows recombination in the former but not in the latter.
This indicates absence of this linkage group in isolate IPO94269.
For IPO3236IPO94269, only markers from IPO323 could be
mapped on this linkage group, and no markers from IPO94269,
confirming that IPO94269 lacks this linkage group. Lines are
drawn between markers that segregated in both populations. Stars
next to the markers for the IPO3236IPO94269 cross indicate
segregation distortion of the markers; * (P,0.05), ** (P,0.01), ***
(P,0.005) or **** (P,0.001).
Found at: doi:10.1371/journal.pone.0005863.s003 (0.03 MB
PDF)
Figure S3 Confirmation of chromosome loss by PCR amplifi-
cation. A. Confirmation of loss of LG 8 and LG 12 by SSR
amplification. Loci ac-0007 (LG 8) and gga-0001 (LG 12) confirm
that these linkage groups are absent in the underlined progeny
isolates from the crosses IPO3236IPO94269 and
IPO3236IPO95052 as neither of the parental alleles are
amplified. Isolates 1158 and 1179 are positive controls and SSR
ag-0003 (LG 2) is a positive PCR control in all duplex reactions. B.
Confirmation of loss of LGs 13, 15, A and C by PCR with primers
developed from DArT marker sequence data in the underlined
progeny isolates derived from crosses between M. graminicola
IPO3236IPO94269 and IPO3236IPO95052. Isolates 1158 and
1179 are positive control isolates, except in LGs C and 13 that
have isolates 1158/2026 and 2032/2033, respectively, as positive
checks. For LG 15* the CABMR_07D07 DArT fragment (129 bp)
was used as a positive PCR control, while for the other linkage
groups DArT fragment AHMR_08O09 (728 bp) was used. C.
Confirmation of loss of LG 8 by PCR with primers developed
from DArT marker sequence data in underlined progeny isolates
derived from crosses between M. graminicola IPO3236IPO94269
and IPO3236IPO95052. This figure is composed of eight panels
that are individually divided by a central marker lane. The left part
of each panel represents the three parental isolates of the mapping
populations (IPO323, IPO94269 and IPO95052), two positive
control isolates (1158/1179), and seven progeny isolates that lack
LG 8. The right part of each panel links to Fig. 2D and represents
the two parental isolates (IPO323 and IPO95052), two twin
Fungal Genome Plasticity
PLoS ONE | www.plosone.org 8 June 2009 | Volume 4 | Issue 6 | e5863
isolates (1103/1126), two mirror isolates (1128/1183) and two
twin isolates that lack LG 8 (2137/2139). In all panels DArT
fragment AHMR_08O09 is the positive control (top band in each
panel, 728 bp, located on LG 15).
Found at: doi:10.1371/journal.pone.0005863.s004 (1.60 MB
PDF)
Table S1 Mycosphaerella graminicola progeny isolates (n = 76)
from the IPO3236IPO94269 in planta cross, that was made on
the susceptible bread wheat cultivar Obelisk, that were used for
hybridization to the DArT arrays.
Found at: doi:10.1371/journal.pone.0005863.s005 (0.05 MB
DOC)
Table S2 Mycosphaerella graminicola progeny isolates (n = 164)
from the IPO3236IPO95052 in planta crosses that were made on
the bread wheat cultivar Obelisk and the durum wheat cultivar
Inbar. Sixteen isolates (gray-shaded) were not used, leaving a total
of 148 that were used in the construction of the genetic linkage
map. The first two numbers indicate the year of isolation and the
next three numbers the order of isolation.
Found at: doi:10.1371/journal.pone.0005863.s006 (0.08 MB
DOC)
Table S3 The adapter and primer oligonucleotide sequences
used for generation of the genomic representation (cloning) from
Mycosphaerella graminicola isolates IPO323 and IPO95052 and
for hybridization to the micro-arrays (genotyping) of parental and
progeny isolates.
Found at: doi:10.1371/journal.pone.0005863.s007 (0.04 MB
DOC)
Table S4 Overview of Mycosphaerella graminicola F1 isolates
that lack one or more linkage groups compared to the parental
isolates IPO323, IPO94269 and IPO95052.
Found at: doi:10.1371/journal.pone.0005863.s008 (0.03 MB
DOC)
Table S5 Primer sequences used to verify the absence of several
linkage groups in some progeny isolates of the two crosses. The
primers were developed using the sequences of the DArT markers
located on these linkage groups.
Found at: doi:10.1371/journal.pone.0005863.s009 (0.07 MB
DOC)
Table S6 Primer sequences used to verify the disomy for linkage
group 1, isolate #51. The primers were developed around InDels
obtained by comparison of BAC-end sequences from parental
isolate IPO94269 with the genome sequence of isolate IPO323.
Found at: doi:10.1371/journal.pone.0005863.s010 (0.04 MB
DOC)
Table S7 Overview of type and number of molecular markers
that were scored in the progeny of the cross between Myco-
sphaerella graminicola isolates IPO323 and IPO94269 before and
after grouping.
Found at: doi:10.1371/journal.pone.0005863.s011 (0.03 MB
DOC)
Table S8 Overview of type and number of molecular markers
that were scored in the progeny of the cross between Myco-
sphaerella graminicola isolates IPO323 and IPO95052 before and
after grouping.
Found at: doi:10.1371/journal.pone.0005863.s012 (0.04 MB
DOC)
Table S9 Identified twin isolates in the two progenies derived
from crosses between either Mycosphaerella graminicola isolates
IPO323 and IPO94269 or IPO323 and IPO95052.
Found at: doi:10.1371/journal.pone.0005863.s013 (0.04 MB
DOC)
Table S10 Scoring tables
Found at: doi:10.1371/journal.pone.0005863.s014 (3.33 MB
XLS)
Table S11 Overview of the number of markers for both crosses.
Mapping was performed using the software package JoinMap 3.0.
Found at: doi:10.1371/journal.pone.0005863.s015 (0.03 MB
DOC)
Table S12 Graphical genotyping
Found at: doi:10.1371/journal.pone.0005863.s016 (5.15 MB
XLS)
Table S13 Alignment of the identified linkage groups in the
Mycosphaerella graminicola IPO3236IPO94269 and
IPO3236IPO95052 mapping populations with the identified
chromosomes in the Mycosphaerella graminicola genome se-
quence.
Found at: doi:10.1371/journal.pone.0005863.s017 (0.05 MB
DOC)
Table S14 DArT and SSR markers that showed translocations
between two genetic linkage maps derived from crosses between
either Mycosphaerella graminicola isolates IPO323 and
IPO94269 or IPO95052.
Found at: doi:10.1371/journal.pone.0005863.s018 (0.03 MB
DOC)
Table S15 Back crosses and intercrosses of M. graminicola
IPO3236IPO94269 progeny isolates with isolates that either lost
or gained specific chromosomes.
Found at: doi:10.1371/journal.pone.0005863.s019 (0.05 MB
DOC)
Acknowledgments
We thank Els C.P. Verstappen for generating the M. graminicola progenies
and Ineke de Vries for helping with the DNA isolations. We gratefully
acknowledge the support in DNA sequencing and analyses of Jim Bristow,
Len Pennacchio and Igor Grigoriev at the US Department of Energy –
Joint Genome Institute and Jane Grimwood at the HudsonAlpha Institute
for Biotechnology.
Author Contributions
Conceived and designed the experiments: AHJW TAJVdL GHJK HJS.
Performed the experiments: AHJW TAJVdL SBM SBW AK. Analyzed the
data: AHJW TAJVdL SBM GHJK HJS. Contributed reagents/materials/
analysis tools: AK. Wrote the paper: AHJW TAJVdL SBG RGFV GHJK
HJS.
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PLoS ONE | www.plosone.org 10 June 2009 | Volume 4 | Issue 6 | e5863
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